Probiotic Sonicates Selectively Induce Mucosal Immune Cells Apoptosis through Ceramide Generation via Neutral Sphingomyelinase

Abstract

BackgroundProbiotics appear to be beneficial in inflammatory bowel disease, but their mechanism of action is incompletely understood. We investigated whether probiotic-derived sphingomyelinase mediates this beneficial effect.Methodology/Principal FindingsNeutral sphingomyelinase (NSMase) activity was measured in sonicates of the probiotic L. brevis (LB) and S. thermophilus (ST) and the non-probiotic E. coli (EC) and E. faecalis (EF). Lamina propria mononuclear cells (LPMC) were obtained from patients with Crohn's disease (CD) and Ulcerative Colitis (UC), and peripheral blood mononuclear cells (PBMC) from healthy volunteers, analysing LPMC and PBMC apoptosis susceptibility, reactive oxygen species (ROS) generation and JNK activation. In some experiments, sonicates were preincubated with GSH or GW4869, a specific NSMase inhibitor. NSMase activity of LB and ST was 10-fold that of EC and EF sonicates. LB and ST sonicates induced significantly more apoptosis of CD and UC than control LPMC, whereas EC and EF sonicates failed to induce apoptosis. Pre-stimulation with anti-CD3/CD28 induced a significant and time-dependent increase in LB-induced apoptosis of LPMC and PBMC. Exposure to LB sonicates resulted in JNK activation and ROS production by LPMC. NSMase activity of LB sonicates was completely abrogated by GW4869, causing a dose-dependent reduction of LB-induced apoptosis. LB and ST selectively induced immune cell apoptosis, an effect dependent on the degree of cell activation and mediated by bacterial NSMase.ConclusionsThese results suggest that induction of immune cell apoptosis is a mechanism of action of some probiotics, and that NSMase-mediated ceramide generation contributes to the therapeutic effects of probiotics.

Hepatocarcinogenesis and Ceramide/Cholesterol Metabolism

“…Although several mammalian SMases have been characterized, neutral sphingomyelinase 2 (NSMase 2, SMPD3) and acid sphingomyelinase (ASMase, SMPD1) are the most extensively studied enzymes in ceramide generation, and the most relevant in cell death Morales et al, ACA-MC 8 regulation and pathology. While NSMase-induced ceramide generation has been described as a critical lipid in inflammatory diseases and oocyte maturation [29,30], ASMase with an optimum pH of 4.5-5.5, has been extensively described as a signaling intermediate in cell death pathways, of particular relevance in the progression of liver diseases [24,27,28,31]. ASMase catalyzes the formation of ceramide from SM primarily within the endo-lysosomal compartment, although it can be secreted extracellularly through Golgi trafficking as a secretory ASMase (S-SMase) [3,4].…”

ASMase is required for chronic alcohol induced hepatic endoplasmic reticulum stress and mitochondrial cholesterol loading

“…In addition to de novo synthesis in the ER, ceramide can be generated by sphingomyelin (SM) hydrolysis by sphingomyelinases (SMases) [14]. Although, the neutral SMase (NSMase) has been shown to induce apoptosis [15, 16], this outcome may be dependent on the topology/sidedness of NSMase-induced ceramide generation [17]. Moreover, acid SMase (ASMase) mediates apoptosis in response to cell death receptors, chemotherapy or ionizing radiation [18–20].…”

Glycosphingolipids and cell death: one aim, many ways

“…Neutral sphingomyelinase (NSMase) and acid sphingomyelinase (ASMase) are the most studied enzymes in ceramide generation, which have been involved in pathophysiological processes and disease. In this regard, NSMase-induced ceramide generation has been described as a critical lipid mediator in inflammatory diseases and X. laevis oocyte maturation [28, 29]. Moreover, ASMase has been characterized as a signaling intermediate in extrinsic cell death pathways and liver diseases [30-36].…”

“…Recent evidence has shown that NSMase cooperates with Bak and Bax to promote the mitochondrial pathway of apoptosis [49]. In addition, bacterial NSMase has been identified as a key component of the therapeutic effects of the probiotic formulation VSL#3 in inflammatory bowel disease due to the selective induction of apoptosis of activated mucosal immune cells, as the beneficial effects of VSL#3 were prevented by NSMase inhibition with GW48469 [28]. …”